Cyclosporine A (CsA), tacrolimus (FK506, Prograf®) and sirolimus(rapamycin) are potent immunosuppressive drugs that inhibit T-lymphocyte proliferation. The action of these drugs is mediated through intracellular proteins called immunophilins. These immunophilins are rotamases (enzymes involved in protein folding).
Sirolimus and Tacrolimus share structural homology, and an inhibitory binding domain on a family of immunophilins, called FK506 binding proteins or FKBPs (Abraham et al., Ann Rev. Immunol. Vol. 14, 483 (1996)). Cyclosporin A binds to and inhibits cyclophilin, another immunophilin. In complex with the binding proteins these drugs inhibit secondary targets that regulate signal transduction pathways and result in inhibition of immune cell cycle progression. These pathways mediate and regulate the desired immunosuppression. These and other factors and pathways also systemically produce the undesirable drug effects through immune and other cell types.
Sirolimus and tacrolimus both interact with FKBP12, one member of the FKBP immunophilins, which is expressed in human blood. The dimers of sirolimus/FKBP12 and tacrolimus/FKBP12 complex with and inhibit separate target molecules. The Sirolimus/FKBP12 dimer target is called Mammalian Target of Rapamycin (mTOR). The Tacrolimus/FKBP12 dimer targets Calcineurin ((Abraham et al., (above); Chung et al., Cell Vol. 69, 1227 (1992)). The Cyclosporin A-cyclophilin dimer and tacrolimus/FKBP12 dimer separately can form a pentamer complex with and inhibit a common target, Calcineurin, a serine-threonine phosphatase.
Binding of the sirolimus/FKBP12 dimer to mTOR inhibits T-cell cell cycle progression. In T-cells Calcineurin/Calcium/Calmodulin bound to either tacrolimus/FKBP12 complex or the Cyclosporin A/cyclophilin complex prevents dephosphorylation of, and thus, reduces activation of several systemic signal transduction molecules, including NFAT which stimulates transcription of the immune modulator interleukin-2 (IL-2). The immunosuppressive effects of these drugs are achieved by the previously described multimeric complexes formed by these drugs with their binding proteins, their targets (enzymes that they inhibit), and other requisite cofactors. Tacrolimus has a narrow therapeutic range, because of this, monitoring of tacrolimus levels in patients undergoing tacrolimus immunosuppressive therapy is a standard practice. Current methods measure total (complexed and uncomplexed) drug concentration in blood. U.S. Pat. No. 6,338,946 relates to methods for manually assaying immunosuppressant drugs (with calcineurin-inhibiting activity) in vitro, i.e., by forming a complex of isolated tacrolimus with exogenous binding components immunosuppressant drug, specific immunophilin involved, bovine calcineurin, calmodulin, calcium), in a solid container and detecting the complex with an anti-calcineurin antibody tagged to a detection system. Other available methods extract tacrolimus from blood samples obtained from patients receiving tacrolimus, and measure the amount of extracted tacrolimus by forming in vitro complexes as described above ((Amstrong et al., Clin. Chemistry Vol. 44, pages 2516-2523 (1998)). These measurements are then compared to demographically determined drug toxicity concentration ranges and used to estimate potential toxic effects.
It has been demonstrated that there is a lack of correlation between total drug concentration and immunosuppression; therefore the measurement of total blood concentration of the drug is not predictive of individual immunosuppression responses. Variability in immunosuppressive drug response has been attributed to the discovery of several factors: inactive metabolites of the parent drugs that cross-react with the specific antibodies for the parent drugs in the assays, active metabolites of the parent drug that do not bind the assay antibody (and are not measured), and in great part to the fact that these assays measure total parent drug in a sample, rather than functional immune-suppressive complexes. It must be kept in mind that tacrolimus, sirolimus, and Cyclosporin A and their active metabolites act as immune-suppressants only when they form multimeric complexes with their particular binding immunophilins and target enzymes involved in immune cell suppression.
Each patient has different blood concentrations of these binding proteins and target components (due to age, gender, race, disease states, etc.). Thus, the ability to form immunosuppressive complexes and the number of complexes formed in the presence of each one of these drugs is uniquely (genetically, and/or environmentally) determined in each individual patient. Therefore, each patient has a unique degree of immune suppressive response, depending in part on the presence and abundance of the components required to form the immune-suppressive complexes.
When patients are treated with these hydrophobic immunosuppressive drugs, some of the drug is bound to functional binding proteins and affects immunosuppressive signal transduction pathways, but a significant fraction of drug is non-specifically bound to proteins, lipids, and membranes, becoming sequestered away from the immune cells where the immunosuppressive action takes place. Measuring total blood concentration of the immunosuppressive drug with currently available methods leads to an overestimation of the amount of functional drug present in blood because these methods measure functionally inactive drug as well drug involved in immunesuppression (Alak, A., Therap. Drug, Monit., Vol. 19, pages 338-351 (1997). Additionally, current methods that measure active metabolites of the parent drug lack correlation between their pharmacological activity and their immunologic cross-reactivities (Amstrong et al., Clin. Chemistry Vol. 44, pages 2516-2523 (1998)). It is also important to mention that current methods measuring total immunosuppressive drugs in a patient's blood sample are used to predict potential toxic effects, not to measure immunosuppressive therapeutic effects.
In view of the above, there is a need for assay methods that permit quantification of the functionally active immunosuppressive complexes, i.e. complexes formed in the patient's own blood. It is envisaged, without being bound to a theory, that through this quantification, an estimate of specific binding proteins and target components, and an estimate of potential immunosuppression before, or during, immunosuppressive therapy, will be provided. Specifically, in patients who had not started immunosuppressive therapy, quantification of the functionally active immunosuppressive complexes could help select the most appropriate drug treatment specific for each patient based on the patient's ability to form active complexes with tacrolimus, sirolimus or cyclosporine A, or combinations thereof, without subjecting the patient to unnecessary toxic drug effects caused by current trial and error drug selection methods. In patients already under immunosuppressive therapy, quantification of theoretical maximal immunosuppressive complexes may allow for a more reliable correlation between the pharmacologically active fraction of drug in blood and the immunosuppression observed in vivo in the patient. By adding saturating amounts of drug to a blood sample of a patient undergoing therapy and comparatively evaluating complexes formed in initial samples and in drug-saturated samples, information is obtained on the potential increase in immunosuppressive drug dosage that will result in increased immunosuppression without risking increased negative effects for the patient. Additionally, methods that permit measurement of the fraction of drug that forms functionally active immunosuppressive complexes can be adapted to allow a quantification of the proportions of complexes formed for each immunosuppressive drug when a patient is undergoing dual therapy.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.